High body temperature increases gut microbiota-dependent host resistance to influenza A virus and SARS-CoV-2 infection

Fever is a common symptom of influenza and coronavirus disease 2019 (COVID-19), yet its physiological role in host resistance to viral infection remains less clear. Here, we demonstrate that exposure of mice to the high ambient temperature of 36 °C increases host resistance to viral pathogens including influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High heat-exposed mice increase basal body temperature over 38 °C to enable more bile acids production in a gut microbiota-dependent manner. The gut microbiota-derived deoxycholic acid (DCA) and its plasma membrane-bound receptor Takeda G-protein-coupled receptor 5 (TGR5) signaling increase host resistance to influenza virus infection by suppressing virus replication and neutrophil-dependent tissue damage. Furthermore, the DCA and its nuclear farnesoid X receptor (FXR) agonist protect Syrian hamsters from lethal SARS-CoV-2 infection. Moreover, we demonstrate that certain bile acids are reduced in the plasma of COVID-19 patients who develop moderate I/II disease compared with the minor severity of illness group. These findings implicate a mechanism by which virus-induced high fever increases host resistance to influenza virus and SARS-CoV-2 in a gut microbiota-dependent manner.


Supplementary Figure 8
Effects of outside temperature on severity of ZIKV infection.
Mice were kept at 4 or 22 °C for 7 d before ZIKV infection and throughout infection. Mice kept at 4 or 22°C were infected intravenously with 1.5×10 7 pfu of ZIKV. Mortality was monitored for 14 days (4 °C, n = 38 mice; 22 °C, n = 20 mice). Data are pooled from three independent experiments. Statistical significance was analysed by log-rank (Mantel-Cox) test.

Supplementary Figure 9
Effects of outside temperature on severity of EMCV infection.

Supplementary Figure 10
High-heat exposure of aged mice had improved survival after influenza virus infection. a, b, One hundred seventeen-to 122-weeks-old mice were kept at 22 or 36 °C for 7 d before influenza virus infection and throughout infection. Mice kept at 22 or 36°C were infected intranasally with 1,000 pfu of influenza virus. Body temperatures (a) and mortality (b) were measured on indicated days after challenge (22 °C, n = 8 mice; 36 °C, n = 8 mice). Data are mean ± s.e.m. Data are representative of two independent experiments. Statistical significance was analysed by two-tailed unpaired Student's t-test (a) or log-rank (Mantel-Cox) test (b). ***P<0.001 (a; 0 d p.i., p=0.0000000248; 4 d p.i., p=0.0000000304; 7 d p.i., p=0.000002735).

Supplementary Figure 12
Analysis of gut microbiota composition in high heat-exposed control, LF-fed, or Abx-treated mice.

Supplementary Figure 14
Effects of outside temperature on the cecal metabolome profiles of mice and hamsters. a, b, PCA plot of the cecal metabolome profiles in mice normalized by Pareto (a) and loading scatter plot (b). c, Box plots indicating cecal amounts of metabolites in mice that had |PC1 coefficient values| > 0.13 in PCA. Significant differences are indicated based on Tukey-Kramer test. d, e, PCA plot of the cecal metabolome profiles in Syrian hamster normalized by unit variance (d) and loading scatter plot (e). f, Box plots indicating cecal amounts of metabolites in Syrian hamster that had PC1 coefficient values < -0.115 in PCA. Statistical significance was analysed by two-tailed unpaired Student's t-test (c; DCA, p=0.002845; Taurine, p=0.000146; Butyrate, p=0.000000009885; f; Cholate, p=0.0121; DCA, p=0.130155). The centre line denotes the median value (50th percentile), while the white box contains the 25th to 75th percentiles of dataset. The black whiskers mark the 5th and 95th percentiles (c, f).

Supplementary Figure 15
The levels of bile acids in serum.

Supplementary Figure 16
The levels of bile acids in Liver.

Supplementary Figure 17
The levels of bile acids in serum of high heat-exposed mice.

Supplementary Figure 18
Bile acids protect mice from influenza virus infection.
a-c, Room temperature-exposed mice given 0.5 mM of UDCA (a, c) or TDCA were infected intranasally with 1,000 pfu of influenza virus. Mortality (a, n = 25 mice for water-fed, n = 30 mice for UDCA-treated; b, n = 16 mice for water-fed, n = 17 mice for TDCA-treated) and virus titer in the lung wash (c; water, n = 14 mice; UDCA, n = 17 mice) were measured on indicated days after challenge. Data are mean ± s.e.m. Data are pooled from two independent experiments. Statistical significance was analysed by log-rank (Mantel-Cox) test (a, b), or twotailed unpaired Student's t-test (c; p=0.016282). *P<0.05.

Supplementary Figure 19
The levels of bile acids in serum of control or CA-treated mice.

Supplementary Figure 21
Bile acids inhibit influenza virus replication. a, Effects of bile acids (BAs) on influenza virus replication. Red arrows indicate enhancement. Blue blunt ended bars indicate inhibition. b-e, MDCK cells were infected with influenza virus in the presence or absence of 125 μM of DCA. Cells were collected at 6 h post infection, and intracellularly stained with nucleoprotein (NP)-specific antibody (b). Percentages of NP-positive cells are shown (c; mock, n = 2; PR8, n = 5; PR8 + DCA, n = 5). Total RNAs were extracted from uninfected or virus-infected cells at 24 h p.i. and influenza virus NP mRNA levels were assessed by quantitative reverse transcription PCR (d; n = 6). Cell-free supernatants were collected at 24 and 48 h p.i. and analyzed for virus titer by standard plaque assay using MDCK cells (e; n = 6). Data are mean ± s.e.m. Data are representative of two independent experiments. Statistical significance was analysed by two-way analysis of variance (ANOVA) test (c, d) or two-tailed unpaired Student's t-test (e). **P<0.01, ***P<0.001 (e; 24 h p.i., p=0.000007746; 48 h p.i., p=0.000000015).

Supplementary Figure 22
Effects of TGR5 and FXR agonists on influenza virus replication. a-c, MDCK cells were infected with influenza virus PR8 (an amantadine-resistant strain) in the presence or absence of indicated amounts of GW 4064, HY-14229, or amantadine. Cell lysates were collected at 24 h p.i. and analyzed by immunoblotting with indicated antibodies (a). Cell-free supernatants were collected at 24 h p.i. and analyzed for virus titer by standard plaque assay using MDCK cells (b; DMSO, n = 4; 100 μM, n = 4; 10 μM, n = 8; 1 μM, n = 8). Total RNAs were extracted from uninfected or virus-infected cells at 24 h p.i. and influenza virus NP mRNA levels were assessed by quantitative reverse transcription PCR (c; n = 6). Data are mean ± s.e.m. Data are representative of two independent experiments (a, c) or are pooled from two independent experiments (b). Statistical significance was analysed by two-way analysis of variance (ANOVA) test (b, c). ***P<0.001, n.s., not significant.

Supplementary Figure 23
Therapeutic effects of HY-14229 on influenza virus replication.
MDCK cells were infected with influenza virus PR8. After infection, the culture medium was replaced with medium with 1 μM of HY-14229 at indicated time points. Cell lysates were collected at 24 h p.i. and analyzed by immunoblotting with indicated antibodies. Data are representative of two independent experiments.

Supplementary Figure 25
Effects of outside temperature on influenza virus-induced cytokine production. a-d, LF-fed, Abx-treated, and control mice kept at 36 °C were infected intranasally with 1,000 pfu of influenza virus. The lung washes were collected at 4 d p.i. and analyzed for CXCL1 by ELISA (a, n = 36 mice for control, n = 15 mice for LF-fed; b, n = 30 mice for control, n = 11 mice for Abx-treated). Leucocytes were isolated from the lung at 7 (c) or 9 (d) d p.i.. The number of Ly6C + Ly6G + neutrophils were analyzed by flow cytometry (c, n = 8 mice for control, n = 8 mice for LF-fed; d, n = 9 mice for control, n = 10 mice for Abxtreated). Data are mean ± s.e.m. Data are pooled from two independent experiments (a, b) or are representative of two independent experiments (c, d). Statistical significance was analysed by two-tailed unpaired Student's t-test. *P<0.05, **P<0.01 (a; p=0.003; b; p=0.0064; c; p=0.01131; d; p=0.01046;).

Supplementary Figure 26
Treatment of bone marrow-derived macrophages with IL-1β stimulates CXCL1 production.
bone marrow-derived macrophages were stimulated with indicated amounts of recombinant mouse IL-1β (rIL-1β). Cell-free supernatants were collected at 24 h post stimulation and analyzed for CXCL1 by ELISA (n = 4). Data are mean ± s.e.m. Data are representative of two independent experiments. Statistical significance was analysed by two-way analysis of variance (ANOVA) test. ***P<0.001.

Supplementary Figure 27
Weight loss and virus titer of SARS-CoV-2-infected hamsters.

Supplementary Figure 28
Body temperature and the levels of bile acids in serum of cold-and room temperature-exposed hamsters.

Supplementary Figure 29
Effects of TGR5 and FXR agonists on SARS-CoV-2 replication.
VeroE6/TMPRSS2 cells were infected with original SARS-CoV-2 (S-614D) in the presence or absence of indicated amounts of GW 4064, HY-14229, or amantadine. Cell lysates were collected at 24 h p.i. and analyzed by immunoblotting with indicated antibodies (n = 2). Data are representative of two independent experiments.